1 Field of the Invention
This invention relates generally to nucleic acid sequences encoding proteins that restore fertility in a plant.
2 Background Art
Considerable effort is being devoted to the development of strategies to increase canola yields due to the importance of canola crops worldwide. One method of obtaining increased yields of canola involves the generation of hybrid canola plants. Due to a phenomenon termed “hybrid vigor” hybrid canola plants are higher yielding than non-hybrid canola plants (Grant, I. and Beversdorf, W., 1985, Can. J. Genet. Cytol. 27:472–478). In fact, manually produced hybrids can yield up to 50% more seed than either of their parental lines. To produce such hybrids on a large scale, however, self-pollination of at least one of the parents of the hybrid cross must be prevented. One means of preventing self-pollination is to incorporate the trait of cytoplasmic male sterility into a seed parent of the hybrid.
Cytoplasmic male sterility (CMS) results in an inability of the plant to produce viable pollen. In some cases, pollen formation is blocked or aborted in a CMS plant because of a gene in a cytoplasmic organelle, the mitochondrion. This widespread and classic non-Mendelian trait results from rearrangements of the mitochondrial genome (Schnable, P. S. and Wise, R. P., 1998, Trends in Plant Sci., 3:175–180). Plants carrying the CMS trait are incapable of self-pollination, and therefore, when a CMS line is planted alongside a male-fertile line, all the seed that forms on the sterile plants is a hybrid of the two parents.
Importantly however, use of the CMS trait in a hybridization scheme produces seeds that are male-sterile since, in most species, the trait is inherited maternally. While the fertility of the resultant seeds is unimportant in some crops (i.e., vegetables), fertility must be restored in the crops for which pollen production is required for formation of the harvested products, as in the case of fruit or seed crops such as canola. In order to restore fertility to the hybrids, specific dominant nuclear genes termed restorers of fertility (Rf) can be introduced into the hybrid plants to suppress the male-sterile phenotype (Schnable, P. S. and Wise, R. P., 1998, Trends in Plant Sci., 3:175–180). Accordingly, the use of CMS for commercial seed production involves the use of three breeding lines, a male-sterile line (female parent), a maintainer line which is isogenic to the male-sterile line but does not contain a sterility inducing mitochondrial genome and a restorer line (male parent).
A crop of particular interest herein is the oilseed crop of the species Brassica napus, commonly referred to as canola. A number of CMS systems have been reported in Brassica species. Five of the systems most commonly used for hybrid seed production are Polima (pol), nap, tournefortii, Kosena and Ogura (ogu). The form of CMS in Brassica napus which is currently thought to be potentially the most useful for hybrid seed production is the ogu system. The ogu system is based on the use of a hybrid cytoplasm in which the male sterility determinant is derived from a radish (Raphanus sativum) cytoplasm. Male sterility induced by ogu cytoplasm is more complete and more temperature stable than any of the other endogenous B. napus CMS systems. Analysis of the ogu mitochondrial genome has indicated that this form of CMS is specified by a novel open reading frame (ORF), orf138, that encodes a polypeptide, ORF138 (Grelon et al., 1994, Mol. Gen. Genet. 243:540–547).
Recently, a Brassica napus restorer line for the ogu system became available (Delourme, R. et al., 1995, Proc. 9th Int. Rapseed Cong. Cambridge, UK 1:6–8). Using this restorer line, it was determined that restoration of fertility resulted in a decrease of the ORF138 protein in stamens as compared to un-restored, ogu sterile lines (Bellaui, M. et al., 1999, Plant Mol. Biol. 40:893–902). However, a drawback to these prior art ogu restorer lines is that hybrids produced using these lines have elevated glucosinolate levels. An elevation of glucosinolate levels in plants is problematic when the plants are used in animal feed because this compound causes digestive problems in animals. Elevated glucosinolate levels are undesirable in canola plants in particular since much of their value is derived from their low levels of glucosinolate compounds.
The elevation of glucosinolate levels results from a dominant gene that is linked to the radish nuclear fertility restorer gene or genes, termed Rfo in the prior art. Rfo, like the ogu cytoplasm, has been introduced from the radish but recombination in the radish chromosomal region surrounding Rfo is suppressed in B. napus (Delourme R. et al., 1998, Theor. Appl. Genet. 97:129–134). Despite considerable effort by several groups, it has not yet been possible to develop stable B. napus lines in which Rfo has been efficiently dissociated from the glucosinolate gene and do not address other deficiencies in Rfo restorer lines, and therefore, the system is not widely implemented.
Accordingly, what are needed in the art are improved lines of canola that can be used as restorers of fertility in hybridization systems. More particularly, it would be beneficial to provide restorer lines of canola containing one or more nuclear fertility restorer genes from Raphanus sativum, which genes are separated from the gene or genes causing increased levels of glucosinolate in the resultant hybrid plants. The present invention also provides a method of using the fertility restorer nucleic acid to select for transgenic plant cells by means of its capacity to restore pollen production to cytoplasmic male sterile plants.